Immunoglobulin adsorption

Abstract

The present disclosure is directed to methods for the preparation of immunoadsorption matrices having IgG molecules adsorbed thereto in a preferred configuration, i.e., adsorbed to the matrix by their (Fc) rather than F(ab) portions. IgG molecules, are selected such that the F(ab) portion of the IgG fraction adsorbed has a more acidic or basic net isoelectric point or pI range than the F(c) end of the molecule, depending on the characteristics of the adsorption surface. For negatively charged surfaces, IgG molecules having relatively alkaline F(c) portions are selected. For positively charged surfaces, IgG with relatively acidic F(c) portions are selected. Additional selection criteria include pI fractionation to provide fractions having well defined pI characteristics as defined by "non-overlap" or "pI range" of F(c) and F(ab) portions pI's. Methods disclosed are particularly well suited to the preparation of colloidal gold immunostains.

what is claimed is: 
     
       1. A method for preparing an adsorption surface by adsorbing thereon selected IgG molecules, the method comprising the steps of: (a) selecting an adsorption surface having a net positive or negative charge;   (b) choosing an IgG species having an F(c) portion with a net pI that is basic with respect to its F(ab) portion in the case of a negatively charged surface, or an F(c) portion with a net pI that is acidic with respect to its F(ab) portion in the case of a positively charged surface;   (c) fractionating IgG moieties of the chosen IgG species to select for a population of IgG molecules having a pI range of less than or equal to approximately 2 pI units and further wherein the net pI of the selected population is acidic, in the case of negatively charged surface, or basic, in the case of a positively charged surface; and   (d) adsorbing the selected IgG population to the selected adsorption surface.   
     
     
       2. The method of claim 1 wherein the selected adsorption surface is a negatively charged surface, the chosen IgG species is rabbit IgG, and step (c) comprises: (a) fractionating the rabbit IgG into fractions according to their relative pI; and   (b) selecting a fraction having a net acidic pI and which exhibits a pI range of less than or equal to 2.   
     
     
       3. The method of claim 2 wherein the selected negatively charged adsorption surface is negatively charged colloidal gold. 
     
     
       4. The method of claim 1 wherein the selected negatively charged adsorption surface is negatively charged nitrocellulose, negatively charged polystyrene, negatively charged colloidal gold, negatively charged colloidal silver, or negatively charged nylon. 
     
     
       5. The method of claim 1 wherein the selected adsorption surface is a positively charged surface, the chosen IgG species is goat, sheep, horse, guinea pig, cow, pig, mouse, rat or human IgG, and step (c) comprises: (a) fractionating the IgG into fractions according to their relative pI; and   (b) selecting a fraction having a net basic pI and which exhibits a pI range of less than or equal to 2.   
     
     
       6. The method of claim 5 wherein the selected positively charged adsorption surface is positively charged colloidal gold, positively charged polystyrene, or positively charged nylon. 
     
     
       7. The method of claim 1 further comprising a step of selecting a pH of adsorption at which adsorption of the F(c) portion of the selected IgG population will be facilitated relative to adsorption of the F(ab) portion and wherein the step (d) is performed at the selected pH. 
     
     
       8. The method of claim 7 wherein selecting a pH of adsorption comprises the steps of: (a) enzymatically digesting an aliquot of the selected IgG population to generate free F(c) and F(ab) portions;   (b) adsorbing the free F(c) and F(ab) portions to the surface at various pH's to determine a pH at which the F(c) portions are preferentially adsorbed relative to the F(ab) portions; and   (c) selecting the pH determined in step (b) for performing the adsorption of the selected IgG population.   
     
     
       9. A method for preparing an adsorption surface by adsorbing thereon selected IgG molecules, the method comprising the steps of: (a) selecting an adsorption surface having a net positive or negative charge;   (b) choosing an IgG species having an F(c) portion with a net pI that is basic with respect to its F(ab) portion in the case of a negatively charged surface, or an F(c) portion with a net pI that is acidic with respect to its F(ab) portion in the case of a positively charged surface;   (c) fractionating IgG molecules of the chosen IgG species to select for a population of IgG molecules wherein the pI values of the F(c) portions of the selected IgG molecules differ and are more basic than the pI values of the F(ab) portions of the selected IgG molecules, in the case of a negatively charged surface, or which differ and are more acidic than the pI values of the F(ab) portions, in the case of a positively charged surface; and   (d) adsorbing the selected IgG population to the selected adsorption surface.   
     
     
       10. The method of claim 9 wherein the net pI of the selected population is acidic, in the case of negatively charged surface, or basic, in the case of a positively charged surface. 
     
     
       11. The method of claim 9 wherein the selected adsorption surface is negatively charged, the chosen IgG species is rabbit IgG, and step (c) comprises: (a) fractionating the IgG into fractions according to their relative pI;   (b) assaying fractions by an aliquot of IgG from the fraction in a manner to generate free F(c) and F(ab) portions;   (c) separating the F(c) and F(ab) portions according to their relative pI's; and   (d) selecting a fraction having IgG molecules with F(c) portions having pI's more basic than the pIs of the F(ab) portion.   
     
     
       12. The method of claim 11 where in the IgG molecules of the selected fraction exhibit a net acidic pI. 
     
     
       13. The method of claim 11 wherein the adsorption surface is negatively charged colloidal gold. 
     
     
       14. The method of claim 11 wherein the selected negatively charged adsorption surface is negatively charged nitrocellulose, negatively charged polystyrene, negatively charged colloidal gold, negatively charged colloidal silver, or negatively charged nylon. 
     
     
       15. The method of claim 9 wherein the selected adsorption surface is positively charged, the chosen IgG species is goat, sheep, horse, guinea pig, cow, pig, mouse, rat or human, and step (c) comprises: (a) fractionating the IgG fractions according to their relative pI's;   (b) assaying fractions by an aliquot of IgG from the fraction in a manner to generate free F(c) and F(ab) portions;   (c) separating the free F(c) and F(ab) portions according to their relative pI's; and   (d) selecting a fraction having IgG molecules with F(c) portions having pI's more acidic than the pI's of the F(ab) portions F(ab).   
     
     
       16. The method of claim 15 wheren the IgG molecules of the selected fraction have a net basic pI. 
     
     
       17. The method of claim 15 wherein the selected positively charged adsorption surface is positively charged colloidal gold, positively charged polystyrene, or positively charged nylon. 
     
     
       18. The method of claim 9 further comprising a step of selecting a pH of adsorption at which adsorption of the F(c) portion of the selected IgG population will be facilitated relative to adsorption of the F(ab) portion and wherein step (d) is performed at the selected pH. 
     
     
       19. A method for adsorbing an immunoglobulin molecule onto a selected adsorption surface comprising the steps of: (a) selecting an adsorption surface having a net positive or negative charge;   (b) choosing an IgG species having an F(c) portion with a net pI that is alkaline with respect to its F(ab) portion in the case of a negatively charged surface, or an F(c) portion that is acidic with respect to its F(ab) portion in the case of a positively charged surface;   (c) selecting a pH of adsorption at which adsorption of the F(c) portion to the surface will be facilitated relative to adsorption of the F(ab) portion; and   (d) adsorbing the IgG to the selected adsorption surface at the selected pH.   
     
     
       20. The method of claim 19 wherein selecting a pH of adsorption comprises the steps of: (a) enzymatically digesting an aliquot of the selected IgG species to generate free F(c) and F(ab) portions;   (b) adsorbing the free F(c) and F(ab) portions to the surface at various pH's to determine a pH at which the F(c) portions are preferentially adsorbed relative to the F(ab) portions; and   (c) selecting the pH detemined in step (b) for performing the adsorption of the selected IgG population.   
     
     
       21. The method of claim 19 wherein the selected adsorption surface is a negatively charged surface and the chosen IgG species is rabbit IgG. 
     
     
       22. The method of claim 20 wherein the adsorption surface is colloidal gold. 
     
     
       23. The method of claim 20 wherein the selected negatively charged adsorption surface is negatively charged nitrocellulose, negatively charged polystyrene, negatively charged colloidal gold, negatively charged colloidal silver, or negatively charged nylon. 
     
     
       24. The method of claim 19 wherein the selected adsorption surface is a postively charged surface and the chosen IgG species is goat, sheep, horse, guinea pig, cow, pig, rat, mluse or human IgG. 
     
     
       25. The method of claim 24 wherein the positively charged adsorption surface is positively charged colloidal gold, positively charged polystyrene, or positively charged nylon.